Alexa flour 594 secondary antibody

The experiments were performed as described previously [25 (link)]. Yeast cells were grown to mid-log phase, shifted for 1 h to their restrictive temperatures and fixed with 4% formaldehyde for 1 h at room temperature. After zymolyase treatment, spheroplasts were transferred to polylysine-coated microscope slides, permeabilized with 0.5% triton X-100/P-solution (0.1 M phosphate-buffer pH6.5, 1.2 M sorbitol), blocked with antibody blocking buffer ABB (1x PBS, 10% heat-inactivated FCS, 0.3% tween-20) for 1 h at room temperature and incubated with anti-Nop1 antibodies diluted 1:2500 in ABB overnight at 4°C. Following four times washing with ABB, anti-rabbit Alexa Flour 594 secondary antibody (from Invitrogen) diluted 1:100 in ABB was added for 2 h at room temperature. After washing with ABB and 0.1% Tween-20/PBS, the DNA was stained with Hoechst 33342 and microscopy studies were performed as described in “Fluorescence in situ hybridizations”.
For co-localization of 25S rRNAs and Nop1 proteins, the in situ hybridization protocol was performed first with the Cy3-labeled oligonucleotide HK2200 against 25S rRNAs followed by the immunofluorescence protocol with anti-Nop1 primary antibodies and anti-rabbit Alex Fluor 488 secondary antibodies (from Invitrogen).

Tumor cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 for 10 min, and then blocked with 5% bovine serum albumin (Amresco, OH, USA). The cells were incubated with the primary LC3-I/II antibody (CST, #4108, 1:100) overnight at 4 °C. After washing with PBS, the cells were incubated with Alexa-flour 594 secondary antibody (Invitrogen) at room temperature for 1 h. Nuclei were stained with DAPI (Sigma) for 5 min. Following three rinses with PBS, the cells were imaged by confocal laser scanning microscopy (FluoView FV1000, Olympus).

At the indicated time point, C2C12 myoblasts were stained with a myosin heavy chain antibody (MF20 in 1:10 dilution) as previously described (le May et al., 2011 (link)). The cells were then washed with 1X PBS and incubated with Alexa Flour^®594 secondary antibody (Invitrogen) and 0.1 μg/ml of Hoechst (Molecular Probes) to stain the DNA. The coverslips were mounted on slides with 50% glycerol. Axiovert 200M microscope, AxioCam HRM camera, and AxioVision Rel 4.8 software (Zeiss) were used to capture the images through a ×10 objective. For each coverslip, five random images were analyzed. ImageJ software was used for cell counting.

Immunofluorescence analysis was performed as described by us earlier [31 (link), 34 (link)]. U87MG cells were plated in a 24-well plate on a coverslip at a density of 0.1 × 10⁶cells/well and allowed to attach overnight followed by treatment with 2.5 μM and 5 μM penfluridol for additional 48 h. The cells were fixed with formalin and Triton-X100 solutions to permeabilized the cells. After blocking with 6% goat serum in 1%BSA, cells were incubated overnight with antibody against OCT4 (1:400). Cells were washed with PBS and again incubated overnight with Actin antibody (1:2000). Next day cells were incubated with AlexaFluor 488 secondary antibody (Invitrogen, Carlsbad, CA) as well as AlexaFlour 594 secondary antibody (Invitrogen, Carlsbad, CA) separately, after washing three times with PBS in between and after incubation. Coverslips were mounted on slides (mounting media with DAPI) and images were taken using multi photon confocal microscope (Nikon).